This proposal is designed to study the mechanism of donor specific allograft tolerance induced through mixed allogeneic bone marrow chimerism in the clinically relevant monkey model. The specific aims of this project include determining what particular cells from the donor bone marrow are necessary to establish immunological tolerance and whether or not long-term chimerism is necessary to maintain tolerance to the allograft. In tolerant animals whose donor derived cells are undetectable in the peripheral blood, lymphoid tissues will be extensively examined for evidence of chimerism. The role of the allograft itself in the maintenance of long-term immunological tolerance will also be studied. Donor bone marrow will be fractionated using magnetic bead separation of cells labeled with mAbs to particular cell lineage markers. The particular bone marrow cell subsets will be transferred into MHC mismatched recipients which have been subjected to the non-lethal preparative regimen to allow for bone marrow engraftment and establishment of mixed chimerism. Lymphoid tissues will then be examined by immunohistochemistry and by PCR to detect donor derived cell lineages at sequential times after bone marrow transfer. Irradiated bone marrow will be transferred at the time of kidney transplant to determine whether the donor bone marrow acts passively as a source of antigen rather than actively in the establishment of tolerance. These studies will have important implications for the understanding of tolerance in primates and may ultimately lead to establishment of clinical protocols to prevent organ transplant rejection across allogeneic barriers without the need for chronic immunosuppression.